Receptors: their identification, characterization, preparation and use

ABSTRACT

Disclosed are novel hormone and hormone-like receptors wherein the trans-activation transcription domain(s) is modified in terms of position and/or copy number or otherwise versus parent receptor, such novel receptors having increased trans-activation transcription activation properties surprisingly superior to the parent receptor. Also disclosed are the recombinant methods and means for preparing such receptors and assays based upon the use of such receptors for screening and identifying putative materials that can affect such receptors and/or for the expression via induced transcription of a reporter or other desired, preferably heterologous gene or DNA product.

ACKNOWLEDGEMENT

This invention was made with Government support under Grant No. GM 26444awarded by the National Institutes of Health. The Government has certainrights in the invention.

RELATED APPLICATIONS

Reference is made to U.S. Pat. application Ser. No. 922,585, filed Oct.24, 1986 and to its continuation-in-part application U.S. Ser. No.108,471, filed Oct. 20, 1987, issued as U.S. Pat. No. 5,071,773,counterparts of which exist as published documents by certainjurisdictions outside of the U.S., and to U.S. Ser. No. 128,331, filedDec. 2, 1987 now abandoned and to its continuation-in-part applicationU.S. Ser. No. 276,536, filed Nov. 30, 1988, now issued as U.S. Pat. No.4,981,784. All of these applications refer in various respects tohormone receptors and compositions thereof, and to methods for theirpreparation, and use, particularly in novel assay systems. The entiredisclosures of each of these applications is hereby incorporated byexpress reference herein.

FIELD OF THE INVENTION

The present invention relates generally to the identification andcharacterization of certain polypeptide sequences that function astranscription trans-activation domains, and to their preparation anduse, particularly in the preparation of novel intracellular hormone orhormone-like members of the steroid hormone superfamily of receptors,for example, steroid receptor polypeptides, thyroid polypeptides andretinoid polypeptides including those of the human species, whereadvantage is provided in terms of trans-activation transcriptioninitiation activity by augmenting the effect of said domains.

More particularly, the present invention is directed to such novelreceptor polypeptides, wherein the transcription trans-activationdomains have been augmented in effect so as to produce novel entitiesthat exhibit increased transcription initiation activity surprisinglysuperior to the parent molecule.

Novel aspects relating to the preparation of such transcriptiontrans-activation molecules, including novel DNA isolates encoding sameand the transcription trans-activation domains, expression vectorsoperatively harboring these DNA sequences and hosts transfected withsaid vectors are included within the scope of this invention.

Most particularly, the present invention concerns the use of the noveltranscription trans-activation hormone or hormone-like receptors of thepresent invention in assays for screening various putative materialsthat may have operative binding affinity for the novel hormone orhormone-like receptors hereof. In a preferred embodiment, this aspect ofthe invention provides an assay for screening such putative materials,for example, steroid agonists and antagonists in an enhanced, so-calledtrans-activation system.

BACKGROUND OF THE INVENTION

The patent applications cited supra disclose, inter alia., thecharacterization and preparation of various hormone and hormone-likereceptors, including steroid, thyroid and retinoid receptors such asthose represented by the glucocorticoid, mineralcorticoid, thyroid,estrogen related and retinoid classes, and specifically, theglucocorticoid, estrogen, aldosterone and retinoic acid receptorsthemselves. These specific receptors have been the subject ofconsiderable research and form the particular bases for the inventionsdisclosed and claimed in these patent applications. Similarly, theextant, parallel scientific literature has focused on the specificreceptors listed above from among the classes of receptors that exist.

It is known, for example, that the glucocorticoid receptor belongs to alarge super-family of ligand-dependent transcription factors that havethemselves diverse roles in homeostasis, growth and development.Comparison of complementary DNAs encoding these receptors, as well asmutational analyses of their coding sequences have identified certainfunctional domains within the molecule that are thought responsiblerespectively for DNA binding, hormone binding and nuclear localization.See Evans, et. al., Science 240, 889 (1988) for a review of this subjectmatter. In the case of the glucocorticoid receptor, the so-called DNAbinding domain spans some sixty-six amino acids and is highly conservedamong various species and this domain has been found to be required inorder to activate transcription. See Hollenberg, et. al., Cell 49, 39(1987), Miesfeld, et. al., Science 236, 423 (1987), Danielsen, et. al.,Mol.Endo 1, 816 (1987), Kumar, et. al., Cell 51, 941 (1987), Gronemeyer,EMBO J. 6, 3985 (1987), and Waterman, et. al., Mol.Endo 2, 14 (1988).This region has been found to contain nine invariant cysteines residuesand although the contribution of each cysteine residue to overallfunction is unknown, as is the actual structure formed by this domain,it has been proposed that these cysteine residues coordinate two zincions to form two DNA binding, so-called finger domains which result in aternary structure thought responsible for its localization and bindingto the requisite DNA site. See Klug, et. al., Tr.Biochem.Sci 12, 464(1987), Bens, et. al., Cell 1 52. 1 (1988), and Evans, supra.

In a location nearer the carboxy-terminal end distal from the DNAbinding region is the so-called ligand binding domain which has thedemonstrated ability to block activity of the receptor in the absence ofhormone. Thus, presence of the requisite hormone relieves the inhibitionof the receptor to activity. Deletion of this region has been found toproduce a hormone-independent transcription activator. See Godowski, et.al., Nature 325, 365 (1987), Hollenberg, et. al., supra, Kumar, et. al.,supra, Danielsen et. al., supra, and Adler et. al., Cell 52, 685 (1988).

In contrast to these two domains, the sequences lying towards theamino-terminal region from the DNA binding domain are poorly understoodboth as to structure, and particularly, function. This region isextremely variable both in size and in composition amongst the variousreceptors--See Evans, supra--and may contribute to the heterogeneity ofreceptor function. See Kumar et. al., supra, and Tora et. al., 333, 185(1988).

Despite extensive analysis, some of which having been reported in thescientific literature, the region(s) that determines trans-activation oftranscription initiation remains poorly characterized. Trans-activationdomains can be defined as polypeptide regions that, when combined withthe DNA binding functional domain, increase productive transcriptioninitiation by RNA polymerases. See Sigler, Nature 333, 210 (1988), Brentet. al., Cell 43, 729 (1985), Hope et. al., Cell 46, 885 (1986), Ma et.al., Cell 48, 847 (1987), Ma et. al., Cell 51, 113 (1987), Lech et. al.,Cell 52, 179 (1988), and Hope et. al., Nature 333, 635 (1988).

Previous research of the human glucocorticoid receptor by linkerscanning mutagenesis identified two regions outside of the DNA bindingregion having a role in transcription activation. These regions weredefined as τ₁ and τ₂. Giguere et. al., Cell 46, 645 (1986). Furtherresearch from these laboratories has also resulted in the report of aco-localization of trans-activation and DNA binding functions. SeeHollenberg et. al., supra. Miesfeld, et. al., supra, Danielsen et. al.,supra, and Waterman et. al., supra. As a composite, this research hasgiven rise merely to an emerging picture of an increasingly modularmolecule with discrete domains, each contributing to the identifiedproperties of ligand-binding, DNA-binding and trans-activation oftranscription. However, until now, the region(s) determining thetrans-activation activity, was not at all well understood. Thus, thepicture based upon existing research lacks an appreciation of thedynamic nature of the steroid receptors and how the various domainsdetermine the cascade of events initiated by ligand-binding andconsummated by promoter-specific trans-activation.

Further, although previous research has identified functional "domains",there has been little systematic effort to identify amino acids thatcontribute to the specific activities of the molecule itself. Thus, theprevious identification of steroid receptor trans-activation regionsresulted only from a demonstrated loss of activity via deletion orinsertional mutagenesis, but in no case have the properties of theregions themselves been confirmed in assays that reflect a dominant gainof function.

Thus, Godowski et. al., Science 241, 812 (1988), report results thatshow that the glucocorticoid receptor binding region and that the seconddomain occupies a region near the receptor amino-terminus. Similarly,Webster et. al., Cell 54, 199 (1988) report on an inducibletranscription activation function of the estrogen and glucocorticoidreceptors, and these researchers speculate that the relative positionsof the hormone regions (i.e., ligand and DNA-binding domains) are notimportant for the transcription induction activity of the receptor. Yet,these researchers admit that they have no definition of the exactlocation and nature of what they call the hormone-inducible activatingdomain, to say nothing of its characterization and role intrans-activating potential.

As a starting point for the present invention, Giguere et. al., supra,demonstrated loss of activity in the glucocorticoid receptor based uponan assay measuring transcription activity, when random site-directedmutagenesis was performed at several locations of the molecule. As afollowup, Hollenberg et. al. deleted regions within the molecule, againdemonstrating overall loss of transcription activity induced by suchremoval of stretches of amino acids.

It is an object of the present invention to identify and characterizethe domain(s) responsible for trans-activation transcription activity,and the characterization of such domain(s) in respect of amino acidcomposition and sequence, to explore the functional interaction of thedomain(s), if any, with both the DNA-binding and ligand-binding domainsof a given receptor, and finally, to exploit such knowledge via themanipulation of such identified and characterized trans-activationtranscription domain(s) so as to increase the overall transcriptionactivity of the given receptor so manipulated.

The present invention thus provides novel hormone or hormone-likereceptors that have been modified by advantage of knowledge of theidentity and characterization of the trans-activation transcriptionactivity domain(s), by modifications thereof so as to produce novel,heterologous receptors that have increased activity compared with theparent molecule. It is an object of the present invention to providenovel, heterologous, optionally hybrid receptors having increasedtrans-activation transcription activity and otherwise having DNA-bindingand ligand-binding domains that may be borrowed from various differentreceptors. It is a further object of the present invention to providenovel assays whereby putative receptor agonists and antagonists can bescreened and evaluated for potential commercial exploitation. See alsoPtashne, Nature 335, 683 (1988).

SUMMARY OF THE INVENTION

The present invention is predicated upon the identification, isolationand characterization of the trans-activation transcription domains ofintracellular hormone or hormone-like receptor polypeptides that has inturn enabled the discriminate characterization of the receptor itself,both in terms of physical attributes and the biological function andeffect of their various domains. This information has in turn enabledthe production of harnessed, recombinant systems useful for preparingthe novel receptors hereof having augmented transcription activationproperties.

It has been determined, based upon the information provided herein, thatreceptors contain trans-activation transcription domains that areposition independent and autonomous in function. Thus, the presentinvention provides for novel hormone or hormone-like receptors whereinthe trans-activation transcription domains are augmented in theirability to activate transcription. Such novel receptors of thisinvention contain trans-activation transcription .domains additional tothe parent molecule, positioned in a manner to provide further increasein transcription activity. These novel receptors may be hybrids whereinthe DNA-binding and the ligand-binding domains are provided fromreceptors of the same or different class and/or species.

The present invention is also directed to the use of such novelreceptors for in vitro bio-assays for determining the functionality of aputative receptor or a putative hormone or hormone-like material.Bio-assays may take the form, for example, of challenging a novelreceptor hereof with one or more of a battery of test materials thathave putative hormone or hormone-like activity and that can potentiallymodulate the bio-function of said receptor and monitoring the effect ofsaid material on said receptor in an in vitro setting.

The present invention is further directed to the preparation of suchnovel receptors hereof via recombinant DNA technology in all relevantaspects, including a DNA molecule that is a recombinant DNA molecule ora cDNA molecule consisting of a sequence encoding said receptor or atrans-activation transcription domain thereof, and to requisiteexpression vectors operatively harboring such DNA comprising expressioncontrol elements operative in the recombinant host selected for theexpression, and to recombinant host cells transfected with suchoperative expression vectors.

The present invention is further directed to a method for inducing theexpression of DNA encoding a reporter molecule or other desiredheterologous polypeptide comprising inducing transcription of the DNAencoding said polypeptide by a complex formed by a novel receptor hereofand a corresponding ligand capable of binding to said receptor, in an invitro setting wherein said receptor and said DNA encoding saidpolypeptide are produced via recombinant expression in a transfectedcell host system.

The present invention thus embraces a hormone or hormone-like receptoras a polypeptide having increased trans-activation transcriptionactivity of a promoter with which it is associated, by virtue of itsintrinsic ability to bind to a DNA sequence response element of saidpromoter or by its ability to associate with other polypeptide(s) thatbind to said DNA sequence response element, and having trans-activationtranscription activity greater than that of its corresponding parentreceptor.

The present invention is directed to recombinant DNA technology in allaspects relating to the use or the characterization of thetrans-activation transcription domain of a hormone or hormone-likereceptor for DNA isolates production, including cross-hybridizable DNAisolates, devising expression vectors therefor, transfected hostsproducing therewith and methods comprising a method of use utilizingsuch information to devise cells or cell lines harboring geneticinformation sufficient for such cells or cell lines to produce suchreceptors such that they can be used as such or in expression systems orin assays for determining the activity of corresponding putativeligands.

DETAILED DESCRIPTION OF THE INVENTION

1. Brief Description of the Drawings

FIG. 1 depicts the identity, and transcription activity, of varioushuman glucocorticoid receptors (hGR) entities hereof that have beenmodified from the parent molecule in the trans-activation transcriptiondomains (tau₁). Wild-type hGR (wt) and tau₁ mutants are schematicallyrepresented. Functional regions are hatched (tau₁), stippled(DNA-binding domain), or indicated by "DEX" (hormone binding domain).Numbers above each receptor define amino acid positions. Heavy verticalbars identify boundaries of an inserted fragment. Relative luciferase(reporter molecule) activity was measured by MTV-LUC using 10⁻⁷ Mdexamethasone (corresponding steroid), except receptors indicated by "C"after the activity which are constitutively active.

FIG. 2 sets forth the luciferase activity of various tau₂ receptorshereof. The wild-type hGR is represented at the top. The tau₂ regionextends from amino acids 526 to 556 and is represented by a solidrectangle. Replacements of the tau region are indicated by a solidrectangle (tau₂ ) or by hatched rectangles for the amphipathic alphahelix ("aah"). Relative luciferase activities were measured by MTV-LUCin the presence of 10⁻⁷ dexamethasone and are followed by "(C)" whenhormone-independent. Asterisks indicate site of the amino acid end oftruncated molecule.

FIG. 3 depicts the construction of hybrid steroidal receptor ofglucocorticoid-thyroid hormone receptors whose trans-activationtranscription activity is increased by the addition of tau: domains. Asegment of the human glucocorticoid receptor cDNA encoding amino acid 77to 262 (encoding the tau₁ domain) was inserted in the rat alpha thyroidhormone receptor cDNA at a position corresponding to amino acid 21, inone or multiple copies. The parental receptor is rTR alpha with a BamHIlinker inserted at the unique BstEII site in the amino terminus.Constructs were transfected into CV-1 cells with TRE-CAT and CATactivity measured in the absence or presence of T₃.

FIG. 4 sets forth the point mutational analysis of the hGR DNA-bindingdomain. The amino acid sequence of the hGR DNA-binding domain is given.Each line represents information believed to be encoded by part of aseparate exon. The consensus sequence (con) for the steroid hormonereceptor super-family is presented below the hGR sequence, withinvariant (bold), conserved(standard type) and non-conserved (dashes)amino acids indicated. Amino acids converted to lysine are topped bycircles. Transcription activity of mutants assayed with MTV-CAT andcompared with hGR-SV are indicated as greater than 10% (filled circles),1% to 10% (half-filled circles), and less than 1% (open circles).

2. General Methods and Definitions

Amino acid identification uses the single- and three-letter alphabets ofamino acids, i.e.:

    ______________________________________                                        Asp   D      Aspartic acid                                                                             Ile   I    Isoleucine                                Thr   T      Threonine   Leu   L    Leucine                                   Ser   S      Serine      Tyr   Y    Tyrosine                                  Glu   E      Glutamic acid                                                                             Phe   F    Phenylalanine                             Pro   P      Proline     His   H    Histidine                                 Gly   G      Glycine     Lys   K    Lysine                                    Ala   A      Alanine     Arg   R    Arginine                                  Cys   C      Cysteine    Trp   W    Tryptophan                                Val   V      Valine      Gln   Q    Glutamine                                 Met   M      Methionine  Asn   N    Asparagine                                ______________________________________                                    

Steroid receptors hereof are prepared 1) having methionine as the firstamino acid (present by virtue of the ATG start signal codon insertion infront of the structural gene) or 2) where the methionine is intra- orextracellularly cleaved, having its ordinarily first amino acid, or 3)together with either its signal polypeptide or conjugated protein otherthan its conventional signal polypeptide, the signal polypeptide or aconjugate being specifically cleavable in an intra- or extracellularenvironment. In all events, the thus produced receptor, in its variousforms, is recovered and purified to a level suitable for intended use.See Supra.

The "hormone or hormone-like receptors" of this invention include thereceptors specifically disclosed, for all species thatcross-hybridization exists, most notably other mammalian receptors, aswell as related (e.g., gene family) receptors of the same orcross-hybridizable species that are enabled by virtue of DNA isolationand characterization and use via cross-hybridization techniques fromsaid specific receptors or from identification via immunocross-reactivity to antibodies raised to determinants in the usualmanner known per se. It also includes functional equivalents of all ofthe above, including interspecies or intraspecies receptors whereinDNA-binding and/or ligand-binding domains are swapped with one another,or otherwise differing in one or more amino acids from the correspondingparent, or in glycosylation and/or phosphorylation patterns, or inbounded conformational structure.

"Expression vector" includes vectors which are capable of expressing DNAsequences contained therein, where such sequences are operatively linkedto other sequences capable of effecting their expression. It is implied,although not always explicitly stated, that these expression vectors maybe replicable in the host organisms either as episomes or as an integralpart of the chromosomal DNA. "Operative," or grammatical equivalents,means that the respective DNA sequences are operational, that is, workfor their intended purposes. In sum, "expression vector" is given afunctional definition, and any DNA sequence which is capable ofeffecting expression of a specified DNA sequence disposed therein isincluded in this term as it is applied to the specified sequence. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of "plasmids" which refer to circular double strandedDNA loops which, in their vector form, are not bound to the chromosome.In the present specification, "plasmid" and "vector" are usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors which serve equivalent functions and which becomeknown in the art subsequently hereto.

Apart from the novelty of the present invention involving themanipulation by means of repositioning or augmentation of thetrans-activation transcription domains of a parent steroid receptor, itwill be understood that the novel steroid receptors of the presentinvention may otherwise permissively differ from the parent steroidreceptor in respect of a difference in one or more amino acids from theparent entity, insofar as such differences do not lead to a destructionin kind of the basic steroid receptor activity or biofunctionality.

"Recombinant host cells" refers to cells which have been transfectedwith vectors constructed using recombinant DNA techniques.

"Extrinsic support medium" includes those known or devised media thatcan support the cells in a growth phase or maintain them in a viablestate such that they can perform their recombinantly harnessed function.See, for example, ATCC Media Handbook, Ed. Cote et. al., American TypeCulture Collection, Rockville, MD (1984). A growth supporting medium formammalian cells, for example, preferably contains a serum supplementsuch as fetal calf serum or other supplementing component commonly usedto facilitate cell growth and division such as hydrolysates of animalmeat or milk, tissue or organ extracts, macerated clots or theirextracts, and so forth. Other suitable medium components include, forexample, transferring, insulin and various metals.

The vectors and methods disclosed herein are suitable for use in hostcells over a wide range of prokaryotic and eukaryotic organisms.

In addition to the above discussion and the various references toexisting literature teachings, reference is made to standard textbooksof molecular biology that contain definitions and methods and means forcarrying out basic techniques encompassed by the present invention. See,for example, Maniatis, et. al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York, 1982 and the various referencescited therein, and in particular, Colowick et. al., Methods inEnzymology Vol 152. Academic Press, Inc. (1987). All of the herein citedpublications are by this reference hereby expressly incorporated herein.

The foregoing description and following experimental details set forththe methodology employed initially by the present researchers inidentifying and characterizing and preparing particular receptors. Theart skilled will recognize that by supplying the present informationincluding the location and makeup of the trans-activationtranscriptional domain of a given receptor and how it can be manipulatedto produce the novel receptors hereof, it is not necessary, or perhapseven scientifically advisable, to repeat these details in theirendeavors to reproduce this work. Instead, they may choose to employalternative, reliable and known methods, for example, they maysynthesize the underlying DNA sequences encoding a particular novelreceptor hereof for deployment within similar or other suitable,operative expression vectors and culture systems. Thus, in addition tosupplying details actually employed, the present disclosure serves toenable reproduction of the specific receptors disclosed and others, andfragments thereof, using means within the skill of the art havingbenefit of the present disclosure. All of such means are included withinthe enablement and scope of the present invention.

3. Detailed Description of Particularly Preferred Embodiments

The present invention was premised upon use of the glucocorticoidreceptor as a model herein for the preparation of novel modifiedentities thereof including hybrids of the glucocorticoid receptor withother receptors such as the thyroid receptor, particularly in theswapping of DNA-binding and ligand-binding domains to make up suchhybrids. In each case the essence of tis invention, namely, therepositioning and/or augmentation of the trans-activation transcriptiondomain(s) so as to create novel receptors whose trans-activationtranscriptional activity is increased over the parent, is illustratedherein using the glucocorticoid receptor as the parent or hybridsthereof upon which comparisons were made for the novel trans-activationtranscription domain modified versions.

It will be understood therefore that for receptors that are known in theart, whether wild-type, hybrids, or functional equivalents as set frothherein, they are suitable as starting materials for the trans-activationtransceiptional domain(s) modifying aspects of the present invention.

4. Examples

The following examples detail materials and methods employed in theexperimental procedures that follow: Point Mutagenesis andTransceiptional Activation

To define systematically the role of the DNA binding domain in thefunction of the receptor, research employed extensive site-directedmutagenesis of this conserved region. By testing the role of individualamino acids this method has the potential to determine whether mutantswhich affect trans-activation are independent from those which influenceDNA binding.

The sequence of the hGR DNA binding region is given in FIG. 4, followedby the consensus sequence for the steroid hormone receptor superfamily.Among members of the superfamily, this domain contains 20 invariant and12 conserved residues. All invariant amino acids, as well as, eightconserved and eight non-conserved residues, were changed to glycine. Theability to stimulate transcription from the GR-responsive MTV promoterand to complex specifically with a glucocorticoid response element (GRE)DNA fragment in vitro was measured.

The steroid-dependent enhancement of transcription from MTV-CAT for eachmutant is given in Table I and is indicated above each altered aminoacid in FIG. 4. All activities are relative to the wild type receptorhGR-SB, which yields an average induction of approximately 1000 fold.Thus, even 1% residual activity is significant. The deletion mutantslisted in Table I, delta420-451 and delta450-487, remove the first andsecond halves of the DNA binding domain, respectively, with the breakpoint corresponding to an exon-exon boundary within the humanmineralcorticoid and chicken progesterone receptor cDNAs. Both have nomeasurable activity indicating that neither "finger" alone is sufficientfor activation.

Of the glycine point mutants, all of those which alter one of the nineinvariant cysteine residues destroy activity, consistent with the ideathat they are critical for function. An additional, non-conservedcysteine at position 431 is not necessary for function. Surprisingly,only five of the remaining 24 glycine mutants are completely inactive.The five non-functional alleles are all invariant residues, suggestingthat they are involved in general aspects of DNA binding, not indetermining sequence specificity. However, not all invariant residuesare critical for function. For example, when aspartic acid 426 isconverted to glycine there is almost no loss in activity. Seven out ofeight mutants with conserved amino acids changed to glycine arefunctional, with half of this group retaining greater than 40% activity.Similarly, mutation of non-conserved amino acids within the DNA bindingdomain in no instance produces a receptor with less than 10% activityand generally causes little reduction in function. Thus, despite somesignificant exceptions, a good correlation exists between theconservation of an amino acid and the extent of functional loss whenconverted to glycine, with all invariant cysteines playing a criticalrole for function.

                  TABLE 1                                                         ______________________________________                                        Comparison of Transcriptional Activity and DNA                                Binding for hGR Glycine Mutants                                                          Relative CAT.sup.a                                                                        Relative DNA Binding.sup.b                             hGR Mutant Activity    in vitro                                               ______________________________________                                        hGR-SB     100         100                                                    Δ420-451                                                                           <1           1                                                     Δ450-487                                                                           <1           1                                                     G421       <1          <1                                                     G423       <1          <1                                                     G424       <1           1                                                     G426        75          75                                                    G431        80          10                                                    G432        10          30                                                    G433        2           1                                                     G437        20          8                                                     G438       <1          <1                                                     G441       <1          <1                                                     G442       <1           60                                                    G444       <1           1                                                     G445       <1          <1                                                     G446        40          6                                                     G447       <1           2                                                     G450        60          50                                                    G453        90          60                                                    G455        80          6                                                     G457       <1           2                                                     G460        55          90                                                    G462        80          40                                                    G463       <1           1                                                     G465        1           1                                                     G467        40          6                                                     G470        3           4                                                     G471        10          6                                                     G473       <1          <1                                                     G474        60          95                                                    G476       <1          <1                                                     G477       <1           1                                                     G479        65          40                                                    G480        90          50                                                    G481       <1           3                                                     G486        10          1                                                     ______________________________________                                         .sup.a Activity was measured with MTVCAT in CV1 cells.                        .sup.b The DNA binding assay is described elsewhere (Hollenberg et al.,       1987). The value given was derived from total immunoprecipitated counts       but reflects specific binding as determined by gel electrophoresis.      

IN VITRO DNA BINDING OF POINT MUTANTS

The CAT activity measured in the transcription assay is the sum ofmultiple individual functions including nuclear localization, DNAbinding, dimerization and perhaps the allosteric events andprotein-protein interactions that ultimately result in activation. Ifmore than one essential function is encoded by the DNA binding domain,some of the non-functional point mutants may still retain their abilityto bind DNA but fail to activate. To explore this possibility, eachmutant protein was produced by transfection of the correspondingexpression vector into COS-1 cells and assayed for its ability, in crudeextracts, to form a specific DNA-protein complex with radiolabeled DNA.The activity of each mutant in this immunoprecipitation DNA bindingassay is given in Table I; the value shown represents specific bindingof the GRE fragment. In general, there is good correspondence betweenthe ability to activate transcription and to bind DNA in vitro. Mutantsfor which DNA binding activity is significantly lower thantranscriptional activity, such as G446, G455, G467 and G486, may beunstable in vitro. The transcriptional activation measured for thesemutants is a result of specific interaction with DNA in vivo, sincedeletion of GREs from the MTV promoter abolishes their activity.

Only a single mutant, G442, which converts the lysine directly followingthe first putative finger to glycine, has lost the capacity toefficiently stimulate transcription while maintaining affinity for DNAin vitro. The specificity of binding to GREs relative to non-specificsequences is only minimally affected, indicating that its dramatic lossin activity is not due to an inability to bind promoter sequences invivo. This is an intriguing mutant because it suggests that DNA bindingis not sufficient for activation. Perhaps G442 prevents a secondaryevent, such as an allosteric change, following the primary protein-DNAinteraction. Despite this exception, these results demonstrate thecysteine-rich 66 amino acid region to embody the DNA binding domain.Indeed, although DNA binding is necessary for trans-activation thisfunction must be located in other regions of the receptor.

The G442 species has particular significance in terms of utility becauseit is the single species prepared that fails to result in activity butstill shows evidence of substantial DNA binding. It is contemplated, forexample, that an assay can be devised exploiting the property of bothG442 and the I550* species. The I550* species lacks completely aligand-binding domain, and as such, is not responsive to the presence orabsence of hormone. The hormone to a specific ligand-binding siterelieves the inhibition of the molecule to act as a trans-activationtranscription factor. Lacking this domain means that the I550* specieswill always produce activity in an assay with or without presence ofsteroid. On the other hand, the G442 species in the same assay willalways be inactive with or without steroid. By devising an appropriateassay where both receptors G442 and I550* are present in the system,initially there will be 100% activity based upon the contribution of theI550* species alone. As the appropriate steroid is added to the system,the activity observed will fall increasingly toward zero with time.Administration of a putative antagonist along with the appropriatesteroid, if active as such, would restore the activity as the antagonistwould interfere with the action of the steroid thus reducing the overallactivity and a rebound in activity would be seen.

If DNA binding and τ functions are truly separable, the possibilityexists to replace the hGR DNA binding domain with a non-receptor DNAbinding domain to produce a hybrid activator protein with a new promoterspecificity. To test this possibility, the hGR cysteine-rich region wassubstituted with the first 74 amino acids of yeast GAL4. These GAL4amino acids are sufficient for sequence-specific DNA recognition, buthave no transcriptional activation capability. The ability totrans-activate lies outside the DNA binding domain and is encoded in twoseparate regions of the protein. Therefore, in order to produce afunctional transcription factor fusions between the GAL4 DNA bindingdomain and hGR must contain trans-activation functions contributed bythe hGR.

To assay function of hGR-GAL4 hybrids, a GAL4-responsive promoter,deltaMTV-GAL-CAT, was constructed from MTV-CAT. This fusion gene wasrendered GR-non-responsive and GAL4-inducible by replacement of GREsequences with a synthetic GAL4 binding site. When measuring theactivation of hGR and GAL4 on this promoter, hGR cannot producemeasurable stimulation from this promoter, whereas GAL4 can increase CATexpression about 20-fold relative to a control expression vector. Thisis consistent with previous reports that GAL4 can function in highereukaryotic cells. The stimulation by GAL4 is clearly mediated throughthe GAL4 synthetic binding site; deletion of this site renders thepromoter non-responsive to GAL4.

Fusions between the GAL4 DNA binding domain and potential hGR activationdomains were examined for their ability to stimulate the GAL4-responsivereporter. To demonstrate that the hGR DNA binding domain is not requiredfor the activity of this hybrid, the GAL4 DNA binding domain wassubstituted for the hGR DNA binding domain to produce the hybrid GgalG.(The hGR, including amino terminus (G-), DNA binding domain (G), andcarboxy terminus (G), is referred to as G-G-G, respectively. Replacementof the hGR DNA binding domain with that of GAL4 generates G-gal-G.) Theresultant hormone-dependent hybrid clearly can function in the absenceof the hGR DNA binding domain and actually acts as a more potenttranscription factor than GAL4 in this assay system, giving a 500 foldincrease in CAT activity with addition of hormone. Unexpectedly, G-gal-Gcan also stimulate delta MTV-CAT without a GAL4 binding site, but onlyabout 5% of the measured on delta MTV-GAL-CAT. The plasmid delta MTV-ATmay contain a cryptic GAL4 recognition site that is revealed only withthe stronger G-gal-G activator and not the weaker GAL4. Indeed,activation is dependent on the GAL4 DNA binding domain; G-gal-G isfunctional on deltaMTV-CAT whereas G-G-G is not.

The trans-activation capability of G-gal-G must be determined by theamino- or carboxy-terminal regions of hGR, since the GAL4 DNA bindingdomain alone is inactive. Accordingly, each of these hGR regions wasindividually tested for its ability to complement the GAL4 DNA bindingfunction. Both hybrids are functional with Ggal(delta) displayingconstitutive activity while (delta)galG is fully hormone dependent.Therefore, autonomous trans-activation functions are embodied in boththe N-terminal and C-terminal segments of the hGR, although subject todifferent constraints.

REARRANGED AND PARTIALLY DUPLICATED HGR MUTANTS

Fusion with the GAL4 DNA binding domain demonstrated the presence ofdistinct trans-activation properties in the hGR amino-terminal 420 aminoacids and carboxy-terminal 300 amino acids. Mutants of the hGR with tau₁duplications were assayed on the luciferase derivative of MTV-CAT,MTV-LUC. Activity values determined with the MTV-luciferase fusion geneand MTV-CAT are equivalent for previously described deletion mutants.FIG. 1 shows the series of τ₁ mutant derivatives and their luciferaseactivity relative to wild type receptor. Absence of τ₁ reduces activityto 5%, while the tandem duplication mutant GR11 acts as a "super"receptor with 310% activity. Mutants GR12, GR14, GR17 and GR18 revealthat can function between the DNA and hormone binding regions, as wellas on the amino terminal side of the DNA binding domain, giving rise ineach case to a hormone-dependent activator (FIG. 1). This indicates aremarkable flexibility of receptor structure. The ability of τ₁ toincrease activity is independent of both the amino and carboxy termini,as shown in FIG. 1 by comparison of GR13 and GR14 activities, and GR15with I515* and GR16. The τ₁ region may not account for alltrans-activation ability in the amino terminus as shown by the 4-foldgreater activity of GR18 relative to GR14. Also in support of thisproposition, deletion of both τ₁ and the carboxy terminus in GR15 leaves1% residual activity, a ten-fold induction, which can be abolished bydeletion of the majority of the amino terminus (compare GR15 and GR26,FIG. 2).

A second region with potential trans-activation character is τ₂, locatedat the very amino terminal end of the hormone binding domain (aminoacids 526-556). This region has five negatively charged residues in astretch of 18 amino acids and is implicated in receptor activity by thetwo-fold difference in activation of truncation mutants 1550* and I515*(FIG. 2). To determine whether τ₂ constitutes an activator domain it wasintroduced adjacent to, or in place of τ₁. FIG. 2 shows that this regionacts to give a 3-fold increase in activity when introduced into theamino terminus independent of τ₁ (compare GR20 with "wt", GR21 withΔ77-262). A second copy gives a further 2-fold increase, so that a pairof τ₂ regions gives an overall increase in activity of six-fold (GR22).Therefore, like τ₁, the position of τ₂ in the receptor is flexible, itsactivity is cumulative and its function can be constitutive (e.g.,GR25).

Constructions similar to the τ₂ mutants GR21 and GR22 were constructedusing the synthetic amphipathic α helix, "aah," containing 20% acidicresidues and demonstrated to possess trans-activation properties in thecontext of yeast GAL4 (Giniger et. al., Nature 330, 670(1987). The sizeand charge characteristics of the "aah" sequence are similar to the τ₂region, which led us to explore its potential activity in the context ofthe hGR. Indeed, a similar increase in activity of mutants with singleor multiple copies of τ₂ and aah is observed (FIG. 3; compare GR21 withGR23, GR22 with GR24), suggesting that these regions may performequivalent functions. These results support and extend the notion of themodular nature of trans-activation domains.

TRANS-ACTIVATION (τ) DOMAINS

We have defined discrete hGR trans-activation regions according to thefollowing two criteria: deletion decreases activity and duplicationincreases it. By these standards two regions of the receptor, of 200 and30 amino acids, encode trans-activation functions. The localization ofthese two regions does not exclude a role for additional activatorsequences within the hGR. Examination of the primary sequences of τ₁ andτ₂ fails to reveal any obvious homology with the exception that bothregions have acidic character. This property is noteworthy becauseactivation domains in the yeast transcription factors GAL4 and GCN4,although lacking obvious sequence identity, are rich in acidic residues.This apparent similarity does not demonstrate that any of the identifiedactivator regions in either GAL4, GCN4, or the glucocorticoid receptorare functioning through a common mechanism, although this seems likely.The potential for a common mechanism is further supported by theobservation that the synthetic amphipathic α helix ("aah") sequence canfunctionally replace τ₂ and to some extent τ₁. The lack of obvioussequence and size relatedness of τ₁ and τ₂ and the yeast activationsequences leads to the view that trans-activation functions might beembodied by the net context of negatively-charged residues on thesurface of the DNA-bound protein.

HGR MODULARITY

The modular nature of the hGR has emerged not only from primary sequencecomparisons within the steroid receptor superfamily, but also by theability to exchange functional domains to create novel chimericactivators. The DNA-binding domain of the hGR has been replaced withthat of the human estrogen receptor (Green et. al., Nature 325, 75 andChambon, (1987) and with the unrelated DNA-binding domain from GAL4. Itsposition is not critical since it can function at the amino terminus. Inaddition, the DNA-binding domain can be placed amino or carboxy-terminalto both τ₁ and τ₂ without compromising function of these domains.Consistent with modular properties, the position of τ₁ and τ₂ is notcritical and their multimerization leads to increased receptor function.Thus, we have been able to generate receptors with activity of up tofour times that of the wild-type receptor or with altered DNA bindingspecificity. Hybrid receptors are still hormonally inducible indicatinga non-specific mechanism whereby the hormone binding domain imposes aligand-dependent effect on the rest of the molecule. Experimental detailand discussion follows:

PLASMID AND POINT MUTANT CONSTRUCTION

Plasmid hGR-SB was generated by recombination at the ClaI site betweenlinker scanning mutants I532 and I403S (Giguere et. al., Cell 46, 645(1986)). I403S was derived from I403 by introduction of the SstI adaptor5' GATCGAGCTCGC 3' into the BamHI site. This hGR derivative is parent toall point mutants and is indistinguishable from wild-type receptor inDNA binding and transcriptional activation. To convert the desired codonof hGR-SB to one encoding glycine, the SstI/BamHI, 400-nucleotidefragment from plasmid hGR-SB was first introduced into M13mp18 to yieldsingle-stranded template. Synthetic oligonucleotides of 13-15 bases werethen used to change the desired codon to GGN by standard techniquesfollowed by re-introduction of the altered SstI/BamHI fragment intohGR-SB. Deletions Δ420-451 and Δ450-487 were generated with longeroligonucleotides (20 mers); amino acid positions given definenon-deleted residues at the deletion junction. Mutant sequences weredirectly determined from double-stranded plasmid DNA using alkalinedenaturation (Hattori et. al., Anal.Biochem 152, 232 (1986)) followed bychain termination of synthetic hGR primers.

Reporter plasmid MTV-CAT and its deletion derivative ΔMTV-CAT weregenerated as follows. The HindIII site of pBLCAT2 (Luckow et.al.,Nucl.Acids Res. 15, 5490 (1987)) was first destroyed to generatepTKCAT-H. The HSV-thymidine-kinase promoter of pTKCAT-H was then excisedby digestion with BamHI/BglII and replaced with the BamHI, MTV-LTRfragment of pMTV-TK (Kuhnel et. al., J.Mol.Biol. 190, 367 (1986)),pLS-190/-181 or pLS-96/-88 (Buetti et. al., J.Mol.Biol. 190, 379 (1986))to generate MTV-CAT, -190/-181 MTV-CAT, or -96/-88MTV-CAT, respectively.ΔMTV-CAT was constructed from -190/-181-and -90/-88 MTV-CAT byrecombination between the HindIII sites of these mutants. A ΔMTV-CAT wasconverted to MTV-GAL-CAT by introduction of the synthetic GAL4 bindingsite "17 MX" (Webster et. al., Cell 52, 169 (1988)) into the uniqueHindIII site. Plasmid MTV-LUC was constructed by conversion of theHindIII site of pSVOA/L-A Δ5' (de Wet et. al., Mol.Cell.Biol. 7 725(1987)) to XhoI and introduction of luciferase coding and SV40polyadenylation sequences from this derivative (generated by BamHIdigestion, Klenow polymerase I end-filling and XhoI digestion) intoXhoI/SmaI-digested MTV-CAT.

G-gal-G was derived from pG525 (Laughon et. al., Mol.Cell.Biol. 4,260(1984)). Primer-directed mutagenesis was used to introduce NotI andXhoI sites at coding-sequence nucleotides -10 to -3 and +223 to +228,respectively. The GAL4 DNA binding domain was excised from thisderivative by digestion with NotI and inserted into pRShGR_(NX) (Giguereet. al., Nature 330, 624(1987)) in place of the endogenous DNA bindingdomain. GgalΔ and ΔgalΔ were produced by digestion of GgalG and ΔgalG,respectively, with XhoI, followed by end-filling and ligation. ΔgalG wasconstructed by introduction of a synthetic oligonucleotide duplex (ΔAN,5═ GTACCACCATGGGGC 3') containing a consensus ribosome binding site(Kozak, Nucl.Acid.Res. 12, 857 (1984)), in place of theamino-terminal-coding, Asp718/NotI fragment of G-gal-G.

Generation of mutants Δ77-262, I515* and Δ9-385 has been described byHollenberg et. al., Cell 49, 39, (1987). τ₁ mutants were constructedusing the BglII/BamHI fragment from I262 (Giguere et. al., Cell 46, 645(1986)), encoding amino acids 77-262 of the hGR. This fragment wasinserted into the BamHI sites of hGR linker scanning mutants I262 andI515 to generate GR11 and GR12, respectively. GR13 was derived frompRShGR_(NX) by replacement of the amino terminal coding sequences withthe ΔAN adaptor described above. GR17 was created by insertion of theamino terminal coding BamHI fragment, generated by recombination betweenlinker scanning mutants I9 and I384 (Giguere et. al., supra,) into theBamHI site of I515. Mutant I550* was produced by recombination betweenthe BamHI sites of mutants I550 and I696, thus shifting the readingframe after amino acid 550. This mutant was incorrectly described asδ532-697 in a previous report (Hollenberg et. al., supra). To create τ₂derivatives, an excisable τ₂ coding-fragment was generated by conversionof hGR nucleotides 1704-1709 and 1800-1805 (Hollenberg et. al., Nature318, 635 (1985)) to BamHI and BglII sites, respectively, usingoligonucleotide-directed mutagenesis. The sequence encoding τ₂ was thenintroduced into the BamHI site of I262 to produce GR20 or in place ofthe BglII/BamHI fragment of I262 to generate GR21. GR23 was created byreplacement of the BglII/BamHI fragment of I262 with a syntheticoligonucleotide duplex (5'GATCT GGAAT TACAA GAGCT GCAGG AACTA CAAGCATTGT TACAA CAGCA AGAG 3') encoding the "aah" sequence (Giniger andPtashne, 1987). Mutants with tandem copies of this sequence and τ₂ (GR24and GR22) were generated by standard techniques (Rosenfeld and Kelly,1986). Double mutant derivatives of constructions described above weregenerated by recombination at the ClaI site: GR14 from GR12 and GR13;GR15 from A 77-262 and I515*; GR16 from GR11 and I515*; GR18 from GR13and GR17; GR25 from GR22 and I515*; GR26 from Δ9-385 and I515*.

IMMUNOPRECIPITATION DNA BINDING

DNA binding was measured as described previously (Hollenberg et. al.,supra). Mutant receptor, obtained in a crude COS-1 cell extract aftertransfection, was incubated with a mixture of radiolabeled DNAfragments, one of which contained GREs. Receptor-DNA complexes wereimmunoprecipitated with receptor-specific antiserum and Staph A, freedof protein, counted Cerenkoy, and then electrophoresed through adenaturing polyacrymide gel to verify specific binding. Totalimmunoprecipitated counts were compared. The presence of mutant hGRprotein in each COS-1 cell extract was confirmed by Western blotanalysis.

TRANSFECTION AND LUCIFERASE ASSAYS

Transfection of CV-1 and COS-1 cells was as described previously(Giguere et. al., and Hollenberg et al., supra) using 5 micrograms ofeach plasmid per 10 cm dish. Luciferase assays were performed asdescribed (de Wet et. al., supra).

CELL CULTURE AND TRANSFECTION

Conditions for growth and transfection of CV-1 (African green monkeykidney) cells were as previously described (Giguere et. al., Cell 46,645 (1986)), except that the calcium phosphate precipitate was left onthe cells for 4-8 hours, at which time the media was changed to DMEMwith 5% T₃ free bovine serum minus or plus,10⁻⁷ M addition of T₃(Sigma). Cells were harvested 36 hours after the (Gorman et.al.,Mol.Cell.Biol. 2, 1044 (1982); Hollenberg et. al., Cell 49, 39(1987)). Typically, 5 μg reporter and 1 μg expression vector werecotransfected, along with 2.5 μg RSV-βgal as a control for transfectionefficiency. Acetylated and non-acetylated forms of [¹⁴ C]chloramphenicolwere separated by thin layer chromatography, excised, and quantitated byliquid scintillation counting in Econofluor (DuPont) with 5% DMSO.β-galactosidase assays were performed as described (Herbomel et. al.,Cell 39, 653 (1984)). CAT activity is expressed as percent conversiondivided by β-galactosidase activity.

CONSTRUCTION OF REPORTER AND EXPRESSION PLASMIDS

Synthetic oligonucleotides corresponding to -169 to -200 of the ratgrowth hormone gene or a palindromic TRE (TCAGGTCATGACCTGA) (Glass et.al., Cell 54, 313 (1988)) were inserted into a linker scanning mutant ofMTV-CAT that has a Hind III site at position -190/-181 (Buetti et. al.,J.Mol.Biol. 190, 379 (1986)) or -190/-88MTV-CAT, which has a Hind IIIsite replacing the nucleotides between -88 and -190. Expression vectorswere constructed for the thyroid hormone receptors by inserting thefull-length cDNAs of pheA12 (Weinberger et. al., Nature 324, 641 (1986))and rbeA12 (Thompson et. al., Science 237, 1610 (1987)) between the KpnIand BamHI sites of the pRS vector (Giguere et. al., Cell 46, 645 (1986)and Nature 330, 124 (1987)).

CONSTRUCTION OF CHIMERIC RECEPTORS.

The construction of hGR_(NX) has been described (Giguere, Nature supra).To construct hTRβ_(NX), the cDNA insert of phe A12 (Weinberger, Nature,supra) was subcloned between the KpnI and BamHI sites of M13mp19 andmutagenized by the method of Kunkel, PNAS 82, 488 (1985). Theoligonucleotide used to create the NotI site changed three amino acids:Asp97 to Arg, Lys98 to Pro, Asp99 to Pro. The oligonucleotide used tocreate the XhoI site changed two amino acids: Thr171 to Leu, Asp172 toGly. The mutant receptor cDNA was then transferred to the expressionvector pRS (Giguere, Cell, Nature supra); hybrids were constructed byexchanging KpnI-NotI, KpnI-XhoI or NotI-XhoI restriction fragmentsbetween RShGR_(NX) and RShTRβ_(NX). RShGR_(NX) has about 75% ofwild-type activity, and RShTR _(NX) has about 60% of wild-type activity.For the addition of τ₁ to rTRα, the unique BstEII site at amino acid 21was changed to a BamHI site by inserting an oligonucleotide adaptor thatencoded a BamHI site flanked by BstEII ends. This allowed the in frameinsertion of a BamHI-BglII fragment encoding amino acids 77-262 of thehGR into this site. ΔTT and ΔGG were constructed by deleting theAsp718-NotI fragment of RShTR _(NX) and RShGR_(NX) respectively andreplacing it with an oligonucleotide adaptor of a consensus ribosomebinding site (Kozak Nucl.Acids Res. 12, 857 (1984)).

Amino acids 77 to 262 of the hGR, called τ₁, were inserted in frameafter amino acid 21 of rTRα in one or multiple copies and the resultinghybrid receptors assayed for trans-activation. Table 2shows thataddition of one τ₁ domain increased activity by at least four-fold,while the presence of multiple such domains further increased activity.This is consistent with the modular nature of this domain, anddemonstrates that the activity of thyroid hormone receptors can beaugmented by the addition of a trans-activation domain from a differentreceptor.

                  TABLE 2                                                         ______________________________________                                        Activity of thyroid hormone receptor/τ.sub.1 hybrids.                     Receptor   # of τ.sub.1 domains                                                                   Relative CAT Activity.sup.a                           ______________________________________                                        RShGR      1              0                                                   RSrTRα-Bm+                                                                         0             100                                                  RSrTR-T.sup.1-1                                                                          1            1430                                                  RSrTR-T.sub.1-2                                                                          2            1140                                                  RSrTR-T.sub.1-3                                                                          3            1960                                                  RSrTR-T.sub.1-4                                                                          4             820                                                  ______________________________________                                         .sup.a CAT activity is relative to the induced activity of RSrTRBm+, whic     is the rat alpha thyroid hormone receptor with the BstEII site at amino       acid 21 changed to a BamHI site.                                         

In certain experiments where the amount of receptor expression vector isincreased from 1 μg to 5 μg, relative CAT activity was shown to increaseseveral-fold.

The foregoing description details specific methods that can be employedto practice the present invention. Having detailed specific methodsinitially used to identify, isolate, characterize, prepare and use thereceptors hereof, and a further disclosure as to specific entities, andsequences thereof, the art skilled will well enough know how to devisealternative reliable methods for arriving at the same information andfor extending this information to other intraspecies and interspeciesrelated receptors. Thus, however detailed the foregoing may appear intext, it should not be construed as limiting the overall scope hereof;rather, the ambit of the present invention is to be governed only by thelawful construction of the appended claims.

We claim:
 1. A method for screening materials in a cellular system andidentifying those materials capable of binding to members of the steroidhormone superfamily of receptors, said method comprising:(a) challenginga member of said steroid hormone superfamily of receptors expressed bysaid cellular system in a form having improved trans-activationtranscription activity, wherein said member of said steroid hormonesuperfamily of receptors contains a plurality of at least one parentalreceptor trans-activation transcription domain selected from the groupconsisting of τ₁, τ₂, and functional fragment(s) thereof, locatedoutside the DNA-biding and ligand-binding domain(s) thereof, (b)monitoring the effect of said test material(s) by measuring the amountof transcription induced by said member of the steroid hormonesuperfamily of receptors, and (c) selecting those materials from thebattery of test materials which alter the trans-activation transcriptionactivity of said member of the steroid hormone superfamily of receptors.2. A cellular system expressing a member of the steroid hormonesuperfamily of receptors having increased trans-activation transcriptionactivity, relative to trans-activation transcription activity ofparental receptor from which said member of the steroid hormonesuperfamily of receptors is derived,wherein said member of the steroidhormone superfamily of receptors contains a plurality of at least oneparental receptor trans-activation transcription domain selected fromthe group consisting of τ₁, τ₂, and functional fragment(s) thereof,located outside the DNA-binding and ligand-binding domain(s) thereof. 3.A cellular system according to claim 2 wherein said member of thesteroid hormone superfamily of receptors is based on the humanglucocorticoid receptor.
 4. A cellular system according to clam 2wherein said member of the steroid hormone superfamily of receptorscontains two of said τ₂ domains.
 5. A cellular system according to claim4 wherein the second of said τ₂ is located adjacent to the first.
 6. Acellular system according to claim 4 wherein the second of said τ₂domains is located in a C-terminal distal location from the first τ₂sequence.
 7. A cellular system according to claim 2 wherein said memberof the steroid hormone superfamily of receptors contains two of said τ₁domains.
 8. A cellular system according to claim 7 wherein the second ofsaid τ₁ domains is located adjacent to the first.
 9. A cellular systemaccording to claim 7 wherein the second of said τ₁ domains is located ina C-terminal distal location from the first τ₁ sequence.
 10. Cellularsystem according to any one of claims 2-9; wherein said member of thesteroid hormone superfamily of receptors if lacking a ligand-bindingdomain.
 11. A cellular system expressing human G442 glucocorticoidreceptor, wherein said receptor is characterized by an inability tostimulate transcription from the GR-responsive MTV promoter, andsubstantial ability to bind specifically with a glucocorticoid responseelement.
 12. A cellular system expressing a member of the steroidhormone superfamily of receptors having trans-activation transcriptiondomains additional to that of parent receptor,wherein saidtrans-activation transcription domains are selected from the groupconsisting of τ₁, τ₂, and functional fragment(s) thereof, wherein saidadditional trans-activation transcription domains are independent ofboth the DNA-binding and ligand-binding domains of parent receptor,wherein said additional trans-activation transcription domains arelocated in a position outside of each of said DNA-binding andligand-binding domains, if present, and wherein said receptor has aDNA-binding biofunctionality.